Insulin-Regulated Srebp-1C and Pck1 Mrna Expression in Primary Hepatocytes from Zucker Fatty but Not Lean Rats Is Affected by Feeding Conditions

Insulin-Regulated Srebp-1c and Pck1 mRNA Expression in Primary Hepatocytes from Zucker Fatty but Not Lean Rats Is Affected by Feeding Conditions

Yan Zhang1, Wei Chen1, Rui Li1, Yang Li1, Yuebin Ge1,2, Guoxun Chen1* 1 Department of Nutrition, University of Tennessee at Knoxville, Knoxville, Tennessee, United States of America, 2 College of Pharmacy, South-Central University for Nationalities, Wuhan, Hubei, China

Abstract Insulin regulates the transcription of genes for hepatic glucose and lipid metabolism. We hypothesized that this action may be impaired in hepatocytes from insulin resistant animals. Primary hepatocytes from insulin sensitive Zucker lean (ZL) and insulin resistant Zucker fatty (ZF) rats in ad libitum or after an overnight fasting were isolated, cultured and treated with insulin and other compounds for analysis of gene expression using real-time PCR. The mRNA levels of one insulin-induced (Srebp-1c) and one insulin-suppressed (Pck1) genes in response to insulin, glucagon, and compactin treatments in hepatocytes from ad libitum ZL and ZF rats were analyzed. Additionally, the effects of insulin and T1317 on their levels in hepatocytes from ad libitum or fasted ZL or ZF rats were compared. The mRNA levels of Srebp-1c, Fas, and Scd1, but not that of Insr, Gck and Pck1, were higher in freshly isolated hepatocytes from ad libitum ZF than that from ZL rats. These patterns of Srebp-1c and Pck1 mRNA levels remained in primary hepatocyte cultured in vitro. Insulin’s ability to regulate Srebp-1c and Pck1 expression was diminished in hepatocytes from ad libitum ZF, but not ZL rats. Glucagon or compactin suppressed Srebp-1c mRNA expression in lean, but not fatty hepatocytes. However, glucagon induced Pck1 mRNA expression similarly in hepatocytes from ad libitum ZL and ZF rats. Insulin caused the same dose-dependent increase of Akt phosphorylation in hepatocytes from ad libitum ZL and ZF rats. It synergized with T1317 to induce Srebp-1c, and suppressed Pck1 mRNA levels in hepatocytes from fasted, but not that from ad libitum ZF rats. We demonstrated that insulin was unable to regulate its downstream genes’ mRNA expression in hepatocytes from ad libitum ZF rats. This impairment can be partially restored in hepatocytes from ZF rats after an overnight fasting, a phenomenon that deserves further investigation.

Citation: Zhang Y, Chen W, Li R, Li Y, Ge Y, et al. (2011) Insulin-Regulated Srebp-1c and Pck1 mRNA Expression in Primary Hepatocytes from Zucker Fatty but Not Lean Rats Is Affected by Feeding Conditions. PLoS ONE 6(6): e21342. doi:10.1371/journal.pone.0021342 Editor: Nai Sum Wong, University of Hong Kong, Hong Kong Received February 21, 2011; Accepted May 26, 2011; Published June 22, 2011 Copyright: ß 2011 Zhang et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by the Scientist Development Grant from American Heart Association (09SDG2140003, to G.C.), a start-up fund from the University of Tennessee at Knoxville (to G.C.), and a research grant from Allen Foundation Inc. (to G.C.). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: [email protected]

Introduction [12] and glucose 6-phosphatase catalytic subunit (G6pc) [7], the first and last steps of gluconeogenesis, respectively. For hepatic lipid The increased rate of metabolic diseases, such as obesity, metabolism, insulin increases the expression levels of sterol diabetes and cardiovascular disease, has become a major public regulatory element binding protein 1c gene (Srebp-1c) [13], a health concern [1,2]. The common characteristic of human member of sterol regulatory element-binding proteins (SREBPs) obesity and type 2 diabetes is insulin resistance [3]. Liver plays a which are critical transcription activators for hepatic cholesterol and critical role in mediating glucose and lipid homeostasis regulated fatty acid biosynthesis, and their homeostasis [14]. In liver of by hormones and nutrients. Factors derived from adipose tissues, SREBP-1c deleted mice [15], the fasting-refeeding cycle no longer such as free fatty acid [4], adipokines [5] and inflammatory appropriately regulated the expression levels of critical lipogenic cytokines [6] have been proposed to be responsible for the hepatic genes such as fatty acid synthase (Fas) and stearoyl-CoA desaturase 1 insulin resistance. (Scd1) [16,17]. When liver is insulin resistant, insulin no longer In liver and hepatocytes, insulin regulates the expression of a suppresses gluconeogenesis, but still stimulates lipogenesis, creating variety of genes responsible for glycolysis, glycogenesis and a vicious cycle that aggravates insulin resistance and ultimately lipogenesis, and inhibits gluconeogenesis [7]. This insulin-regulated contributes to the onset of overt diabetes. The co-existence of hepatic gene expression, at least in part, is responsible for glucose hepatic insulin resistance (elevated gluconeogenesis) and sensitivity and lipid homeostasis [8,9]. When liver is insulin sensitive, insulin (elevated lipogenesis) at gene expression level has been observed in induces glycolysis, lipogenesis and suppresses gluconeogenesis in rodent diabetic models [3,8]. However, the mechanism of this co- hepatocytes. For hepatic glucose metabolism, insulin increases the existence of insulin sensitivity and resistance has not been revealed expression of glucokinase gene (Gck) [10,11], the enzyme responsible [18]. for the first step of hepatic glycolysis. It suppresses the expression of The binding of insulin to its receptor initiates a cascade of signal the cytosolic form of phosphoenolpyruvate carboxykinase (Pck1) transduction events that lead to metabolic changes in its target

PLoS ONE | www.plosone.org 1 June 2011 | Volume 6 | Issue 6 | e21342 Impaired Response of Fatty Hepatocytes to Insulin tissues [19]. The two well studied pathways activated by insulin with carbon dioxide. A catheter was inserted into portal vein and stimulation are the phosphatidylinositol 3-kinase (PI3K)–AKT/ connected to a peristaltic pump with liver perfusion medium and protein kinase B (PKB) pathway [20] and mitogen-activated liver digestive buffer (Invitrogen). The inferior vena cava was cut protein kinase (MAPK) pathway [21]. Recently, insulin regulated open to allow the outflow of the media at flow rate of 10 ml/min. Srebp-1c expression has been shown to be mediated by atypical After completion of the digestion, livers were excised from the rat protein kinase C (PKC) [22,23] and mammalian target of and put into a tissue culture plate containing liver digest buffer for rapamycin complex (mTORC) 1 [24]. Elevated activity of PKCf removing connection tissues and allowing the release of hepato- in Goto–Kakizaki type 2 diabetic rats has been attributed to the cytes. Medium containing hepatocytes were filtered through a cell excessive expression of hepatic Srebp-1c [25]. However, how these strainer and spun at 50 g for 3 minutes. The cell pellets were signaling components cause the transcriptional changes remains to washed twice with DMEM containing 5% fetal bovine serum, 100 be elucidated. units/ml sodium penicillin, and 100 mg/ml streptomycin sulfate. Zucker fatty (ZF) rats [26] and its sub strain Zucker diabetic After wash, the isolated hepatocytes were plated onto 60-mm fatty rats [27] have been widely used as rat models for the collagen type I coated dishes (2 to 3 million cells/dish) and development of metabolic diseases [28,29] due to a missense incubated in 4 ml of the same medium at 37uC and 5% CO2. mutation in the extracellular domain of all leptin receptor isoforms After incubation for 3–4 hours, the attached cells were washed [30–32]. Insulin resistance in Zucker fatty or diabetic fatty rats has once with 4 ml of PBS, and incubated in medium A (medium 199 been associated with higher basal insulin secretion caused by with 100 nM dexamethasone, 100 nM 3,39,5-triiodo-L-thyronine increased fuel metabolism in pancreatic beta cells [33,34]. The (T3), 100 units/ml penicillin, and 100 mg/ml streptomycin sulfate) defects in pancreatic beta cell gene expression in Zucker diabetic containing 1 nM insulin for 14–16 hours until being used for the fatty rats [35,36], but not in obese ZF rats who have normal indicated experiments. For the treatments, primary hepatocytes glycemia [37], have been attributed to the development of were washed once with 3 ml of PBS and then incubated in 2 ml of diabetes. However, the insulin-regulated gene expression in medium A containing indicated reagents for indicated time as hepatocytes from these insulin resistant animals has not been shown in the figure legends. studied. Recently, in an attempt to understand how insulin induces RNA extraction and Quantitative Real-Time PCR transcription of its responsive gene, we identified insulin responsive Methods for preparation and analysis of RNA were described elements in the Srebp-1c promoter as two liver X receptor (LXR) previously [41]. The real time PCR primer sets for detecting Fas binding sites and one sterol regulatory element [38]. This suggests (from Dr. Bruce Spigelman’s group), Gck, Pck1, and Srebp-1c [41] that insulin regulates the expression of its responsive genes after it have been published. The primer sets for Insr (forward 59- stimulates the synthesis of endogenous agonists for nuclear CTGGAGAACTGCTCGGTCATT-39, and reverse 59-GGCC- receptor activation. It has been reported that the hepatic ATAGACACGGAAAAGAAG-39), and Scd1 (forward 59- AAGA- expression of Srebp-1c was elevated in liver of Zucker diabetic TATCCACGACCCCAGCTA-39, and reverse 59- TGCAG- fatty rats [39]. We hypothesize that insulin-regulated expression of CAGGGCCATGAG-39) were designed using Primer Express genes involved in glucose and lipid metabolism may be altered in software (Applied Biosystems). The gene expression level was liver of insulin resistant animals. To focus on insulin resistance and normalized to that of 36B4 unless described otherwise. Data were obesity, but not diabetes, we analyzed insulin-regulated gene presented as either the fold induction calculated from the DDCt expression in hepatocytes from ZF rats, which have hyperlipide- values [40] or the difference of the cycle threshold (DCt) numbers mia, but normal glycemia [37]. Herein, we report the regulation of between the experimental gene and 36B4, the invariable control the mRNA levels of Srebp-1c and Pck1, two representative insulin- gene [41]. regulated genes, in hepatocytes isolated from Zucker lean (ZL) and ZF rats. Immunoblot analysis After indicated treatments in figure legends, primary hepatocytes Materials and Methods in a 60 mm dish were washed once with 3 ml PBS and scrapped from the dish in 400 ml of whole-cell lysis buffer (1% Triton X-100, Reagents 10% glycerol, 1.0% IGEPAL CA-630, 50 mM Hepes, 100 mM The reagents for primary hepatocyte isolation and culture have NaF, 10 mM EDTA, 5 mM Sodium orthovanadate, 1.9 mg/ml been published [40]. Reagents for cDNA synthesis and real-time aprotinin, 5 mg/ml leupeptin, 1 mM Benzamide, 2.5 mM DMSF, PCR were obtained from Applied Biosystems (Foster city, CA). pH 8.0). The lysates were allowed to sit on ice for at least Antibodies to phospho-Akt (Thr473) phospho-Akt (Ser473), and 20 minutes before subjected to 200006 g centrifugation for total Akt, were obtained from Cell Signaling Technologies 20 minutes. The protein content in the supernatant was determined (Danvers, MA). All other compounds were purchased from Sigma with PIERCE BCA protein assay kit (Rockford, IL). Proteins (Saint Louis, MO) unless described otherwise. (30 mg/lane) in whole cell lysates were separated on SDS/PAGE, transferred to BIO-RAD Immun-Blot PVDF membrane (Hercules, Animals CA), and detected with primary antibodies according to the Male ZL and ZF rats were bred at UTK or purchased from protocols provided by the manufacturers. Bound primary antibod- Harlan Breeders (Indianapolis, IN). Rats were housed in colony ies were visualized by chemiluminescence (ECL Western Blotting cages, and fed a standard rodent diet before isolation of primary Substrate; Thermo Scientific) using a 1:5,000 dilution of goat anti- hepatocytes. All procedures were approved by the Institutional rabbit IgG (Upstate) conjugated to horseradish peroxidase. Filters Animal Care and Use Committee at the University of Tennessee were exposed to X-ray films (Phenix Research Products, Candler, at Knoxville (Protocol number 1642). NC) for protein band detection.

Hepatocyte isolation and treatments Statistics For primary hepatocyte isolation, ZL or ZF rats in ad libitum or Data were presented as means 6 SD. The number of fasted overnight as indicated in the figure legends were euthanized experiments represented the independent experiments using

PLoS ONE | www.plosone.org 2 June 2011 | Volume 6 | Issue 6 | e21342 Impaired Response of Fatty Hepatocytes to Insulin hepatocytes isolated from different animals on different days. Levene’s test was used to determine homogeneity of variance among groups using SPSS 19.0 statistical software and where necessary natural log transformation was performed before analysis. Multiple comparisons were analyzed by one-way ANOVA. The Independent-Samples T-Test was used to compare two conditions. Differences were considered statistically significant at P,0.05.

Results Elevated mRNA levels of Srebp-1c, Fas, Scd1, but not Insr, Gck and Pck1 in isolated primary hepatocytes from ad libitum ZF rats To analyze the mRNA levels of hepatic insulin-regulated genes, primary hepatocytes were isolated from ZL and ZF rats in ad libitum condition. The mRNA levels of the representative control and insulin-regulated genes involved in hepatic glucose and lipid metabolism were subjected to real-time PCR analysis as shown in Figure 1. The mRNA level of insulin receptor gene (Fig. 1A, Insr) in freshly isolated primary hepatocytes of ZF rats was not significantly different from that of ZL rats. The mRNA levels of three lipogenic genes, Srebp-1c (Fig. 1B), Fas (Fig. 1C) and Scd1 (Fig. 1D) in the freshly isolated primary hepatocytes of ZF rats were significantly higher than those of ZL rats. On the other hand, the mRNA levels of two genes for glucose metabolism, Gck (Fig. 1E) and Pck1 (Fig. 1F), in the freshly isolated primary hepatocytes of Figure 1. The mRNA levels of Insr (A), Srebp-1c (B), Fas (C), Scd1 (D), Gck (E), and Pck1 (F) in freshly isolated hepatocytes from ad ZF rats were similar to those of ZL rats. All these results libitum Zucker lean and fatty rats. The primary hepatocytes were demonstrated that ZF rat hepatocytes had significantly higher isolated from Zucker lean and fatty rats in ad lib. The total RNA was mRNA levels of Srebp-1c, Fas, and Scd1 than ZL rat hepatocytes extracted and subjected to real-time PCR analysis. Results were did, but not that of Insr, Gck, and Pck1. presented as means 6 SD of 2DCt (against 36B4) from five different hepatocyte isolations from lean or fatty rats (* all P,0.05, for comparing the 2DCt values of the indicated transcripts in hepatocytes from lean The changes of Srebp-1c and Pck1 mRNA levels in rats with those from fatty rats using independent-samples t test). response to insulin stimulation were diminished in doi:10.1371/journal.pone.0021342.g001 hepatocytes from ad libitum ZF rats To investigate insulin effects on gene expression in hepatocytes Pck1 mRNA level in fatty hepatocytes at any insulin concentration from ZL and ZF rats, we examined the regulation of the mRNA tested was significantly higher than that in lean hepatocytes at the levels of two representative genes, one insulin-induced gene (Srebp- corresponding insulin dosage. All these results demonstrated that 1c) and one insulin-suppressed (Pck1) gene, which are robustly insulin-mediated regulation of Srebp-1c and Pck1 mRNA expression regulated by insulin at transcriptional level in liver and primary were significantly diminished or impaired in hepatocytes from fatty hepatocytes [24]. Figure 2A shows the 2DCt numbers of Srebp-1c rats in ad libitum. and Pck1 of the control groups in lean and fatty hepatocytes after the overnight pretreatment. The Srebp-1c mRNA level in fatty The primary hepatocytes from ad libitum ZF rats lost hepatocytes was higher than that in lean hepatocytes (25.261.5 vs glucagon-suppressed Srebp-1c mRNA expression, but 28.161.4). However, Pck1 mRNA in lean hepatocytes was not retained the glucagon-induced Pck1 mRNA expression significantly different from that in fatty hepatocytes (22.361.3 vs To examine the responses of hepatocytes to other hormones, 21.760.7). These results demonstrated that the primary lean and hepatocytes from either lean or fatty rats in ad libitum were treated fatty hepatocytes after overnight pretreatment in culture still with glucagon, an islet derived hormone antagonizing insulin maintained similar expression patterns as the freshly isolated action [42]. Figure 3A shows that insulin induced the Srebp-1c hepatocytes did as shown in Figure 1. mRNA expression in lean hepatocytes as anticipated. Glucagon at To compare the mRNA levels of Srebp-1c and Pck1 in lean and 10 nM reduced basal and 100 nM insulin-induced Srebp-1c fatty hepatocytes after insulin stimulation, their expression levels in mRNA expression to 0.760.04- and 1.260.4-fold of the control hepatocytes incubated in increasing concentrations of insulin were value, respectively. However, Srebp-1c mRNA expression in fatty determined as shown in Figure 2 B and C. Figure 2B shows that hepatocytes was not affected by insulin, glucagon or insulin+glu- the Srebp-1c mRNA level in hepatocytes from ad libitum ZL rats was cagon treatment. Figure 3B shows that the Pck1 mRNA expression significantly induced by insulin at the lowest concentration tested in lean hepatocytes was significantly suppressed in the presence of (0.1 nM). However, insulin failed to induce Srebp-1c mRNA insulin. Glucagon dramatically induced Pck1 mRNA expression to expression in hepatocytes from ad libitum fatty rats. Figure 2C 2163.7- and 8.261.7-fold of the control value in the absence and shows that insulin as low as 0.1 nM was sufficient to significantly presence of insulin, respectively. In fatty hepatocytes, insulin at reduce the Pck1 mRNA level in lean hepatocytes (0.460.23-fold) 100 nM still significantly suppressed Pck1 mRNA expression. with further suppression at higher insulin concentrations (Fig. 2C). However, the remaining Pck1 mRNA in fatty hepatocytes was still In contrast, no significant reduction of Pck1 expression can be higher than that in lean hepatocytes (0.460.1- vs 0.0460.01- fold observed until insulin reached 100 nM in fatty hepatocytes. The of the control value). Despite the diminished response of Pck1

PLoS ONE | www.plosone.org 3 June 2011 | Volume 6 | Issue 6 | e21342 Impaired Response of Fatty Hepatocytes to Insulin

mRNA expression to insulin-mediated suppression in fatty hepatocytes, glucagon dramatically induced its expression by 2765.2- and 10.262.3-fold of the control value in the absence or presence of insulin, respectively. The induction folds of Pck1 mRNA mediated by glucagon treatment in fatty hepatocytes were not significantly different from those in lean hepatocytes without or with insulin. These results again demonstrated impairment of insulin-regulated Srebp-1c and Pck1 mRNA expression in hepatocytes from ZF rats in ad libitum. However, only the glucagon- mediated reduction of Srebp1-c mRNA expression, but not induction of Pck1, was diminished in the same cells. In addition, the presence of insulin significantly attenuated glucagon-induced Pck1 mRNA expression in hepatocytes derived from either lean or fatty rats in ad libitum.

Blocking de novo cholesterol biosynthesis suppressed basal and insulin-induced Srebp-1c mRNA expression in hepatocytes from ZL, but not ZF rats in ad libitum To assess the effects of endogenous cholesterol synthesis on the Srebp-1c mRNA expression, hepatocytes from ZL and ZF rats in ad libitum were treated without or with insulin in the absence or presence of compactin, an inhibitor of 3-hydroxy-3-methylglu- taryl-coenzyme A (HMG CoA) reductase, which has been shown

Figure 2. The maintenance of Srebp-1c and Pck1 mRNA expression patterns in primary hepatocyte culture (A), and the responses of Srebp-1c (B) and Pck1 (C) expression to insulin in cultured hepatocytes from ad libitum Zucker lean or fatty rats. Primary hepatocytes were isolated and pre-treated as described in Materials and Methods before they were incubated in medium A without or with increasing concentrations of insulin (0.01 to 100 nM) for 6 hours. Total RNA was extracted and subjected to real-time PCR Figure 3. The effects of insulin and glucagon on the mRNA analysis. A. The expression levels of Srebp-1c and Pck1 in control levels of Srebp-1c (A) and Pck1 (B) in hepatocytes from ad libitum hepatocytes were expressed as 2DCt. B and C. The expression levels of Zucker lean and fatty rats. Primary hepatocytes after pretreatment Srebp-1c (B) and Pck1 (C) in hepatocytes treated with vehicle control as described in Materials and Methods were incubated in medium A was arbitrarily assigned a value of one for its corresponding cell type. without or with 10 nM glucagon in the absence or presence of 100 nM Results were plotted as fold induction and presented as means 6 SD of insulin for 6 hours. Total RNA was extracted and subjected to real-time five independent hepatocyte isolations for both lean and fatty rats PCR analysis. The expression level of the indicated transcripts in the (n = 5 for hepatocyte isolations; * for comparing the 2DCt values of lean control hepatocytes (lean or fatty) was arbitrarily assigned a value of hepatocytes with fatty hepatocytes using independent-samples t test; one for its corresponding cell type. Results were plotted as fold ** for comparing fold induction values of the indicated transcripts in induction and presented as means 6 SD of five independent lean hepatocytes with that in fatty hepatocytes at indicated insulin hepatocyte isolations for both lean and fatty rats (n = 5 for hepatocyte concentrations using independent-samples t test; for Srebp-1c,c.b/a, isolations; for Srebp-1c,b.a.c, b.d, b.b9, and c9.c, using one-way d.a, using one-way ANOVA; for Pck1,e.f, e.h, using one-way ANOVA; ANOVA; for Pck1,g.h.e.f, g9.h9.e9.f9, and e9.e, using one-way all P,0.05,). ANOVA; all P,0.05). doi:10.1371/journal.pone.0021342.g002 doi:10.1371/journal.pone.0021342.g003

PLoS ONE | www.plosone.org 4 June 2011 | Volume 6 | Issue 6 | e21342 Impaired Response of Fatty Hepatocytes to Insulin to suppress the synthesis of endogenous ligands for LXR activation types of hepatocytes reached plateau at 10 and 1 nM insulin, [43]. Figure 4A shows that insulin induced Srebp-1c mRNA respectively. There was no obvious difference of Akt phosphor- expression in lean, but not fatty hepatocytes, supporting results ylation on these two sites upon insulin stimulation for 10 minutes shown in Figures 2 and 3. Compactin at 50 mM was sufficient to in hepatocytes from ZL and ZF rats in ad libitum. To exclude the significantly suppress both basal and insulin-induced Srebp-1c variations of Immunoblot performed in different days, the mRNA expression in lean hepatocytes to 0.660.1- and 1.160.5- phosphorylation of Akt on Ser473 in three control groups or fold of the control value, respectively. However, it did not 1 nM insulin treatment groups of hepatocytes from either ZL or significantly affect Srebp-1c mRNA expression in fatty hepatocytes ZF rats in ad libitum were compared side by side. As shown in without or with insulin. Compactin did not affect Pck1 mRNA Figure 5B, the levels of Akt phosphorylation on Ser473 in lean expression in hepatocytes from either lean or fatty ad libitum rats in hepatocytes were similar to that in fatty hepatocytes from ad libitum the absence or presence of insulin. These results demonstrated that rats. There was no difference of total Akt protein levels in compactin treatment only inhibited basal and insulin-induced hepatocytes from lean and fatty rats without or with insulin. These Srebp-1c mRNA expression in lean, but not that of fatty results indicated that insulin-induced phosphorylation of Akt on hepatocytes. Thr308 or Ser473 was not impaired in hepatocytes from ad libitum ZF rats. Insulin-induced phosphorylation of Akt was not altered in hepatocytes from ZF rats in ad libitum Fasting restored the induction of Srebp-1c, and As the first step to investigate insulin signal transduction suppression of Pck1 mRNA expression mediated by pathway in hepatocytes from ZL and ZF rats in ad libitum, the insulin and T1317 in hepatocytes from ZF rats levels of phosphorylated and total Akt in them after insulin To explore the effects of feeding condition on the insulin- stimulation for 10 minutes was compared. As shown in Figure 5A, regulated gene expression, the mRNA levels of Srebp-1c and Pck1 in insulin started to induce noticeable phosphorylation of Akt on response to insulin stimulation was analyzed in hepatocytes from Threonine 308 (Thr308) and Serine 473 (Ser473) as low as 0.01 ZL and ZF rats in ad libitum or after an overnight fasting. and 0.001 nM in hepatocytes from either ad libitum ZL or ZF rats, Hepatocytes were treated with insulin, T1317 and insulin+T1317, respectively. The phosphorylation of Thr308 and Ser473 in both and the expression levels of Srebp-1c and Pck1 mRNA were examined as shown in Figure 6. Figure 6A shows that insulin or

Figure 5. Immunoblot analysis of phospho-Akt and total Akt levels in primary hepatocyes treated with increasing concentrations (A) or 1 nM (B) of insulin. After overnight pretreatment, primary hepatocytes from ad libitum ZL and ZF rats were incubated in medium A containing indicated concentrations of insulin for 10 min- Figure 4. The effects of compactin and insulin on the mRNA utes. After which, hepatocytes were washed once with 3 ml PBS and levels of Srebp-1c (A) and Pck1 (B) in hepatocytes from ad libitum lysed as described in Materials and Methods. Total protein (30 mg/lane) Zucker lean and fatty rats. Primary hepatocytes after pretreatment was separated on 8% SDS/PAGE gels, detected by specific primary as described in Materials and Methods were incubated in medium A antibodies as indicated, and visualized by chemiluminescence. A. The without or with 50 mM compactin in the absence or presence of 1 nM levels of phoshpo-Akt(Thr308), phosphor-Akt(Ser473), and total Akt in insulin for 6 hours. Total RNA was extracted and subjected to real-time ad libitum lean and fatty hepatocytes treated with increasing PCR analysis. The expression level of the indicated transcripts in the concentration of insulin (0 to 100 nM). Graph was the representative control hepatocytes (lean or fatty) was arbitrarily assigned a value of of three independent experiments with similar results using hepato- one for its corresponding cell type. Results were plotted as fold cytes isolated from three different ad libitum ZL or ZF rats in different induction and presented as means 6 SD of four independent days. B. The levels of phospho-Akt(Ser473) and total Akt in ad libitum hepatocyte isolations for both lean and fatty rats (n = 4 for hepatocyte lean and fatty hepatocytes treated without or with 1 nM insulin. Graph isolations; for Srebp-1c,b.a.c, b.d and b.b9, using one-way ANOVA; represented three independent isolations of hepatocytes from different for Pck1, e/g.f, and f9.f, using one-way ANOVA; all P,0.05). ad libitum ZL or ZF rats in different days, which were run side by side. doi:10.1371/journal.pone.0021342.g004 doi:10.1371/journal.pone.0021342.g005

PLoS ONE | www.plosone.org 5 June 2011 | Volume 6 | Issue 6 | e21342 Impaired Response of Fatty Hepatocytes to Insulin

T1317 alone induced Srebp-1c mRNA expression in hepatocytes from either ad libitum or fasted ZL rats. T1317 synergized with insulin to induce Srebp-1c mRNA expression dramatically. The induction folds of Srebp-1c mRNA mediated by T1317, but not T1317+Insulin, were significantly higher in hepatocytes isolated from fasted lean rats than that that from ad libitum lean rats (37.265.1- vs 12.762.6-fold). In hepatocytes isolated from ad libitum fatty rats, only insulin+T1317, but not insulin or T1317 significantly induced Srebp-1c mRNA expression. In hepatocytes isolated from fasted fatty rats, insulin, T1317 and insulin+T1317 significantly induced Srebp-1c mRNA expression to 14.968.2-, 8.764.1-, and 80616.6- fold of the control value, respectively. These numbers were significantly higher than those of the corresponding groups in hepatocytes from ad libitum rats. The induction folds mediated by T1317 and T1317+insulin in hepatocytes from fasted ZF rats were still lower than those of the corresponding treatments in hepatocytes from fasted ZL rats. These results indicated that overnight fasting partially corrected the impairment of insulin-induced Srebp-1c mRNA expression in hepatocytes from ZF rats in ad libitum. Figure 6B shows the Pck1 mRNA expression levels in hepatocytes from ZL or ZF rats in ad libitum or an overnight fasting treated without or with T1317 in the absence or presence of insulin. T1317 at 1 mM did not affect the Pck1 mRNA expression in hepatocytes from either ad libitum or fasted lean or Figure 6. The mRNA levels of Srebp-1c (A) and Pck1 (B) in fatty rats in the absence or presence of insulin. Insulin at 1 nM response to insulin, T1317 and insulin+T1317 treatments in dramatically reduced the expression of Pck1 mRNA in hepatocytes hepatocytes from ZL or ZF rats in ad libitum or after fasting for from ad libitum lean rats to the same extent as that in hepatocytes overnight. Primary hepatocytes from rats on different feeding conditions were isolated and pre-treated as described in Materials from fasted lean rats. However, it only suppressed Pck1 mRNA and Methods. Hepatocytes were incubated in medium A without or expression in hepatocytes from fasted fatty rats, but not that from with 1 mM T1317 in the absence or presence of 1 nM insulin for 6 hours. ad libitum fatty rats. Insulin+T1317 significantly reduced the Pck1 Total RNA was extracted and subjected to real-time PCR analysis. The mRNA expression levels in hepatocytes from ad libitum lean, fasted expression level of the indicated transcripts in the control hepatocytes lean and fasted fatty, but not ad libitum fatty rats. Insulin and from lean or fatty rats in ad lib or overnight fasting was arbitrarily insulin+T1317 significantly reduced the Pck1 mRNA expression assigned a value of one for its corresponding cell type and feeding condition. Results were plotted as fold induction and presented as levels in hepatocytes from fasted fatty rats to 0.1760.08- and means 6 SD of indicated numbers (in parenthesis after animal) of 0.1560.08-fold of the control group value, which are still independent hepatocyte isolations for both lean and fatty rats (for respectively higher than 0.0460.008- and 0.0460.015-fold (the Srebp-1c:d.b.c.a, g.a/f, j.h.i.a, and m.k/l.a, b.e, c.f, d.g, corresponding groups) in hepatocytes from fasted lean rats, h.b, h.k, j.m, k.e, l.f, and m.g, using one-way ANOVA; for Pck1: indicating partial improvement of insulin action in hepatocytes a9/b9.c9/d9,a9/h9.i9/j9,a9/k9.l9/m9,f9.c9,g9.d9,f9.l9, and g9.m9, from fasted fatty rats. These results demonstrated that an using one-way ANOVA; all P,0.05). doi:10.1371/journal.pone.0021342.g006 overnight fasting of ZF rats restored the insulin-mediated suppression of Pck1 mRNA expression in their hepatocytes. Srebp-1c mRNA expression at all the concentrations tested. Insulin at Discussion 100 nM suppressed Pck1 mRNA expression in fatty hepatocytes. However, the degree of reduction in fatty hepaotcytes was not In the current study, we observed that the mRNA levels of Srebp- comparable to that in lean hepatocytes (Figure 2). This indicates 1c, Fas and Scd1, but not that of Insr, Gck and Pck1, were higher in that insulin-suppressed Pck1 mRNA expression was not completely freshly isolated hepatocytes from ad libitum ZF rats than those from diminished in fatty hepatocytes, not as insulin-induced Srebp-1c was. ad libitum ZL rats. It has been reported that ZF rats had It has been shown that hepatocytes from the hyperinsulinemic ZF hyperinsulinemia, and elevated free fatty acid levels, but normal rats had insulin binding equivalent to that of lean littermates and plasma glucose levels in basal stage or after a glucose load [44]. had no reduction of insulin receptor at 10 weeks of age [48]. Elevated expression of hepatic lipogenic genes have been observed Therefore, low insulin-binding or receptor expression may not be in ZF rats [45] and Zucker diabetic fatty rats [39,46]. Our results the reason for the diminished insulin-regulated Srebp-1c and Pck1 matched these original observations. It has been shown that mRNA expression. This conclusion is supported by the insulin- hepatocytes isolated from rats in different nutritional conditions mediated Akt phosphorylation, which is similar in hepatocytes retained their differences in glycogen deposition which is isolated from ad libitum lean and fatty rats (Figure 5). influenced by the nutritional state [47]. The hepatocytes from ad Insulin regulates the expression of hepatic genes involved in libitum fatty rats still retained the expression patterns of Srebp-1c glucose and lipid metabolism [7,8]. It induces Gck and suppresses and Pck1 after overnight pre-treatment (Figure 2A). These results Pck1 [7], both involved in hepatic glucose metabolism. It also indicated that our current experimental settings retained the induces the expression of Srebp-1c mRNA and in turn, increases characteristics of the hepatocytes in vivo. hepatic fatty acid biosynthesis [13]. If the hyperinsulinemia resulted We have shown here that insulin induced Srebp-1c and suppressed in the elevated mRNA levels of Srebp-1c, Fas and Scd1 in hepatocytes Pck1 mRNA expression in hepatocytes from ad libitum lean rats. In from ad libitum fatty rats, the question becomes why it did not cause hepatocytes from ad libitum fatty rats, insulin no longer induced elevation of Gck and reduction of Pck1 mRNA expression. It seems

PLoS ONE | www.plosone.org 6 June 2011 | Volume 6 | Issue 6 | e21342 Impaired Response of Fatty Hepatocytes to Insulin that the common insulin signaling pathways branched at some significantly induce Srebp-1c mRNA expression in hepatocytes points that will specifically determine the responsiveness of a gene to from ad libitum fatty rats (Figure 5A) suggests that the transcription insulin in a certain condition. Indeed, bifurcation of insulin pathway complex at Srebp-1c promoter in fatty hepatocytes was not sensitive at mTORC1 step has been observed in rat liver which separated the to changes of ligand for LXR activation as it was in lean insulin-derived signals responsible for elevation of Srebp-1c mRNA hepatocytes. It seems that Srebp-1c mRNA expression was locked in from those for reduction of Pck1 mRNA [24]. Whether any a stage which no dynamic change was allowed. This might have branching point is responsible for the phenomenon observed here caused the unresponsiveness of Srebp-1c mRNA in fatty hepato- remains to be investigated. cytes to positive and negative regulatory signals derived from The diminished regulation of mRNA levels of Srebp-1c and Pck1 insulin and glucagon, respectively. Whether there is any change of in response to insulin suggested that hepatocytes from ad libitum the activities of those transcription complexes on Srebp-1c and Pck1 fatty rats might have lost responses to other hormones. Glucagon, promoter in fatty hepatocytes deserves further investigation. a pancreatic hormone antagonizing insulin action [42], has been As caloric restriction has been shown to increase insulin shown to inhibit Srebp-1c and induce Pck1 mRNA expression in sensitivity in human and animals [52–54], the ZL and ZF rats hepatocytes in the absence or presence of insulin [49]. Therefore, were fasted for overnight and their hepatocytes were isolated for glucagon was used to treat hepatocytes from ad libitum lean or fatty measurement of their responses to insulin and T1317. In rats. Glucagon inhibited basal and insulin-induced Srebp-1c mRNA hepatocytes from ad libitum or fasted lean rats, T1317 synergized expression in lean, but not in fatty hepatocytes from ad libitum rats. with insulin to induce Srebp-1c mRNA expression (Figure 6). Only When the Pck1 mRNA expression was analyzed, glucagon induced T1317 alone caused higher induction of Srebp-1c mRNA its expression in both lean and fatty hepatocytes to the same extent expression in hepatocytes from fasted lean rats than that from ad without or with insulin. These results demonstrated that in fatty libitum lean rats, demonstrating limited effects of fasting on insulin- hepatocytes, Pck1 mRNA expression was still responsive to regulated gene expression in hepatocytes from lean rats. However, glucagon stimulation. It is noteworthy that insulin attenuated in hepatocytes from fasted fatty rats, insulin induced Srebp-1c and glucagon-mediated induction of Pck1 mRNA expression in fatty suppressed Pck1 mRNA expression as it did in hepatocytes from hepatocytes to the same degree as that in lean hepatocytes lean rats. In addition, T1317 also induced Srebp-1c mRNA (Figure 3B). It appears that part of insulin signaling system expression in hepatocytes from fasted fatty rats. The Srebp-1c responsible for regulation of Srebp-1c and Pck1 mRNA expression mRNA levels induced by insulin+T1317 in hepatocytes from was impaired in fatty hepatocytes, whereas other parts responsible fasted fatty rats were similar to those from ad libitum lean rats, for attenuation of glucagon action probably remained unchanged. significantly lower than those from fasted lean rats, and The results obtained from insulin dose-response curves of Akt significantly higher than those from ad libitum fatty rats. These phosphorylation (Figure 5) supported this conclusion. Insulin dose- results demonstrated the partial restoration of insulin-regulated dependently phosphorylated Akt on Thr308 and Ser473 to the gene expression in hepatocytes from fasted fatty rats. It has been same extent in hepatocytes from ad libitum ZL or ZF rats. These shown that an overnight fast was sufficient to mobilize fatty acid results indicated that activation of Akt by insulin remains the same from adipose tissues of fatty rats [37]. In addition, fasting has been in lean and fatty hepatocytes. Components of insulin signal shown to partially restore insulin actions on adipocytes from transduction pathways other than Akt may be responsible for the insulin resistant ZF rats [55]. The restoration of insulin-regulated impaired response of Srebp-1c mRNA expression to insulin in fatty Srebp-1c and Pck1 mRNA expression in hepatocytes from fasting hepatocytes. It has been shown that elevation of PKCf activity fatty rat indicates that short term fasting was sufficient to modify contributed to the increased hepatic Srebp-1c expression in type 2 insulin action on hepatocytes from fatty rats. The molecular diabetic rats [22,23] [25]. In addition, insulin-induced Srebp-1c mechanisms that led to the restoration of insulin action in fatty mRNA expression in primary rat hepatocytes requires mTORC1 hepatocytes deserve further investigation. [24]. Whether any of these plays a role in the impairment of Srebp- insulin-mediated induction of Srebp-1c mRNA in primary hepato- In summary, we have demonstrated that insulin-regulated cytes from ad libitum fatty rats remains to be investigated. 1c and Pck1 mRNA expression was diminished in hepatocytes from As insulin induces Srebp-1c transcription via activation of liver X ad libitum fatty rats. This impairment was not due to any change of receptor, a nuclear receptor activated by cholesterol derivatives Akt phosphorylation by insulin in hepatocytes from ZF rats. This is [50], the elevation of its mRNA expression in fatty hepatocytes the first time that the impairment of insulin action was shown at could be caused by the excessive synthesis of endogenous agonists the regulation of mRNA levels in ZF hepatocytes. The fact that a of LXR activation. However, when the endogenous cholesterol simple overnight fasting partially restored insulin-regulated gene biosynthesis was inhibited by HMG CoA reductase inhibitor expression in fatty hepatocytes indicates the existence of potential compactin [43], the Srebp-1c mRNA expression in fatty hepato- pathway which can reverse the insulin resistance in hepatocytes cytes was not suppressed by it as it was in lean hepatocytes from Zucker fatty rats. The understanding of the underlying (Figure 4). This suggests that the excessive synthesis of endogenous molecular mechanisms will help us to combat metabolic diseases. ligands for LXR activation may not be the reason for unresponsiveness of Srebp-1c mRNA expression to insulin in fatty Author Contributions hepatocytes. Alternatively, another ligand not derived from Conceived and designed the experiments: GC. Performed the experiments: cholesterol might have activated LXR. The fact that T1317 GC WC RL YL YG YZ. Analyzed the data: GC. Contributed reagents/ alone, a synthetic ligand for LXR activation [51], could not materials/analysis tools: GC WC RL YL YG YZ. Wrote the paper: GC.

References 1. Mokdad AH, Serdula MK, Dietz WH, Bowman BA, Marks JS, et al. (1999) The 3. McGarry JD (2002) Banting Lecture 2001: Dysregulation of Fatty Acid Spread of the Obesity Epidemic in the United States, 1991–1998. JAMA 282: Metabolism in the Etiology of Type 2 Diabetes. Diabetes 51: 7–18. 1519–1522. 4. Kabir M, Catalano KJ, Ananthnarayan S, Kim SP, Van Citters GW, et al. 2. Must A, Spadano J, Coakley EH, Field AE, Colditz G, et al. (1999) The (2005) Molecular evidence supporting the portal theory: a causative link between Disease Burden Associated With Overweight and Obesity. JAMA 282: visceral adiposity and hepatic insulin resistance. Am J Physiol Endocrinol Metab 1523–1529. 288: E454–E461.

PLoS ONE | www.plosone.org 7 June 2011 | Volume 6 | Issue 6 | e21342 Impaired Response of Fatty Hepatocytes to Insulin

5. Banerjee RR, Rangwala SM, Shapiro JS, Rich AS, Rhoades B, et al. (2004) of a Missense Mutation in Zucker Fatty (fa/fa) Rats. Biochemical and Regulation of Fasted Blood Glucose by Resistin. Science 303: 1195–1198. Biophysical Research Communications 225: 75–83. 6. Shoelson SE, Lee J, Goldfine AB (2006) Inflammation and insulin resistance. 33. Milburn JL, Hirose H, Lee YH, Nagasawa Y, Ogawa A, et al. (1995) Pancreatic J Clin Invest 116: 1793–1801. beta-Cells in Obesity. J Biol Chem 270: 1295–1299. 7. O’Brien RM, Granner DK (1996) Regulation of gene expression by insulin. 34. Zhou YP, Cockburn BN, Pugh W, Polonsky KS (1999) Basal insulin Physiol Rev 76: 1109–1161. hypersecretion in insulin-resistant Zucker diabetic and Zucker fatty rats: Role 8. Shimomura I, Matsuda M, Hammer RE, Bashmakov Y, Brown MS, et al. of enhanced fuel metabolism. Metabolism 48: 857–864. (2000) Decreased IRS-2 and Increased SREBP-1c Lead to Mixed Insulin 35. Garnett KE, Chapman P, Chambers JA, Waddell ID, Boam DSW (2005) Resistance and Sensitivity in Livers of Lipodystrophic and ob/ob Mice. Differential gene expression between Zucker Fatty rats and Zucker Diabetic Molecular Cell 6: 77–86. Fatty rats: a potential role for the immediate-early gene Egr-1 in regulation of 9. Spiegelman BM, Flier JS (2001) Obesity and the Regulation of Energy Balance. beta cell proliferation. J Mol Endocrinol 35: 13–25. Cell 104: 531–543. 36. Griffen SC, Wang J, German MS (2001) A Genetic Defect in ß-Cell Gene 10. Iynedjian PB (1993) Mammalian glucokinase and its gene. Biochem J 293: 1–16. Expression Segregates Independently From the fa Locus in the ZDF Rat. 11. Magnuson MA, Andreone TL, Printz RL, Koch S, Granner DK (1989) Rat Diabetes 50: 63–68. Glucokinase Gene: Structure and Regulation by Insulin. PNAS 86: 4838–4842. 37. Zucker LM (1972) Fat mobilization in vitro and in vivo in the genetically obese 12. Hanson RW, Patel YM (1997) Regulation of Phosphoenolpyruvate Carbox- Zucker rat ‘‘fatty’’. J Lipid Res 13: 234–243. ykinase (GTP) Gene expression. Annual Review of Biochemistry 66: 581–611. 38. Chen G, Liang G, Ou J, Goldstein JL, Brown MS (2004) Central role for liver X 13. Shimomura I, Bashmakov Y, Ikemoto S, Horton JD, Brown MS, et al. (1999) receptor in insulin-mediated activation of Srebp-1c transcription and stimulation Insulin selectively increases SREBP-1c mRNA in the livers of rats with of fatty acid synthesis in liver. PNAS 101: 11245–11250. streptozotocin-induced diabetes. PNAS 96: 13656–13661. 39. Kakuma T, Lee Y, Higa M, Wang Zw, Pan W, et al. (2000) Leptin, troglitazone, 14. Horton JD, Goldstein JL, Brown MS (2002) SREBPs: activators of the complete and the expression of sterol regulatory element binding proteins in liver and program of cholesterol and fatty acid synthesis in the liver. J Clin Invest 109: pancreatic islets. Proceedings of the National Academy of Sciences of the United 1125–1131. States of America 97: 8536–8541. 15. Liang G, Yang J, Horton JD, Hammer RE, Goldstein JL, et al. (2002) 40. Chen G (2007) Liver lipid molecules induce PEPCK-C gene transcription and Diminished Hepatic Response to Fasting/Refeeding and Liver X Receptor attenuate insulin action. Biochemical and Biophysical Research Communica- Agonists in Mice with Selective Deficiency of Sterol Regulatory Element-binding tions 361: 805–810. Protein-1c. J Biol Chem 277: 9520–9528. 41. Chen G, Zhang Y, Lu D, Li N, Ross AC (2009) Retinoids synergize with insulin 16. Flowers MT, Ntambi JM (2009) Stearoyl-CoA desaturase and its relation to to induce hepatic Gck expression. Biochem J 419: 645–653. high-carbohydrate diets and obesity. Biochimica et Biophysica Acta (BBA) - 42. Starke A, Imamura T, Unger RH (1987) Relationship of glucagon suppression Molecular and Cell Biology of Lipids 1791: 85–91. by insulin and somatostatin to the ambient glucose concentration. J Clin Invest 17. Horton JD (2008) PHYSIOLOGY: Unfolding Lipid Metabolism. Science 320: 79: 20–24. 1433–1434. 43. DeBose-Boyd RA, Ou J, Goldstein JL, Brown MS (2001) Expression of sterol 18. Brown MS, Goldstein JL (2008) Selective versus Total Insulin Resistance: A regulatory element-binding protein 1c (SREBP-1c) mRNA in rat hepatoma cells Pathogenic Paradox. Cell Metabolism 7: 95–96. requires endogenous LXR ligands. Proceedings of the National Academy of 19. Taniguchi CM, Emanuelli B, Kahn CR (2006) Critical nodes in signalling Sciences 98: 1477–1482. pathways: insights into insulin action. Nat Rev Mol Cell Biol 7: 85–96. 44. Zucker LM, Antoniades HN (1972) Insulin and Obesity in the Zucker 20. Manning BD, Cantley LC (2007) AKT/PKB Signaling: Navigating Down- Genetically Obese Rat ‘‘Fatty’’. Endocrinology 90: 1320–1330. stream. Cell 129: 1261–1274. ´ 21. Raman M, Chen W, Cobb MH (2007) Differential regulation and properties of 45. Buque´ X, Martınez MJ, Cano A, Miquilena-Colina ME, et al. (2010) A subset of MAPKs. Oncogene 26: 3100–3112. dysregulated metabolic and survival genes is associated with severity of hepatic 22. Matsumoto M, Ogawa W, Akimoto K, Inoue H, Miyake K, et al. (2003) PKCl steatosis in obese Zucker rats. J Lipid Res 51: 500–513. in liver mediates insulin-induced SREBP-1c expression and determines both 46. Chirieac DV, Collins HL, Cianci J, Sparks JD, Sparks CE (2004) Altered hepatic lipid content and overall insulin sensitivity. J Clin Invest 112: 935–944. triglyceride-rich lipoprotein production in Zucker diabetic fatty rats. American 23. Taniguchi CM, Kondo T, Sajan M, Luo J, Bronson R, et al. (2006) Divergent Journal of Physiology - Endocrinology And Metabolism 287: E42–E49. regulation of hepatic glucose and lipid metabolism by phosphoinositide 3-kinase 47. Agius L, Peak M, Alberti KG (1990) Regulation of glycogen synthesis from via Akt and PKC[lambda]/[zeta]. Cell Metabolism 3: 343–353. glucose and gluconeogenic precursors by insulin in periportal and perivenous rat 24. Li S, Brown MS, Goldstein JL (2010) Bifurcation of insulin signaling pathway in hepatocytes. Biochem J 266: 91–102. rat liver: mTORC1 required for stimulation of lipogenesis, but not inhibition of 48. Clark JB, lark CM (1982) Age-Related Changes in Insulin Receptor Regulation gluconeogenesis. PNAS 107: 3441–3446. in Liver Membranes from Zucker Fatty Rats. Endocrinology 111: 964–969. 25. Sajan M, Standaert M, Rivas J, Miura A, Kanoh Y, et al. (2009) Role of atypical 49. Foretz M, Pacot C, Dugail I, Lemarchand P, Guichard C, et al. (1999) ADD1/ protein kinase C in activation of sterol regulatory element binding protein-1c SREBP-1c Is Required in the Activation of Hepatic Lipogenic Gene Expression and nuclear factor kappa B (NFkB) in liver of rodents used as a model of by Glucose. Mol Cell Biol 19: 3760–3768. diabetes, and relationships to hyperlipidaemia and insulin resistance. Diabeto- 50. Janowski BA, Willy PJ, Devi TR, Falck JR, Mangelsdorf DJ (1996) An oxysterol logia 52: 1197–1207. signalling pathway mediated by the nuclear receptor LXR[alpha]. Nature 383: 26. Zucker LM, Zucker TF (1961) Fatty, a new mutation in the rat. Journal of 728–731. Heredity 52: 275–278. 51. Repa JJ, Liang G, Ou J, Bashmakov Y, Lobaccaro JM, et al. (2000) Regulation 27. Clark JB, Palmer CJ, Shaw WN (1983) The diabetic Zucker fatty rat. Proc Soc of mouse sterol regulatory element-binding protein-1c gene (SREBP-1c) by Exp Biol Med 173: 68–75. oxysterol receptors, LXRa and LXRb. Genes & Development 14: 2819–2830. 28. Aleixandre de Artin˜ano A, Miguel Castro M (2009) Experimental rat models to 52. Henry RR, Scheaffer L, Olefesky JM (1985) Glycemic Effects of Intensive study the metabolic syndrome. British Journal of Nutrition 102: 1246–1253. Caloric Restriction and Isocaloric Refeeding in Noninsulin-Dependent Diabetes 29. Unger RH (1997) How obesity causes diabetes in Zucker diabetic fatty rats. Mellitus. J Clin Endocrinol Metab 61: 917–925. Trends in Endocrinology and Metabolism 8: 276–282. 53. Okauchi N, Mizuno A, Yoshimoto S, Zhu M, Sano T, et al. (1995) Is caloric 30. Iida M, Murakami T, Ishida K, Mizuno A, Kuwajima M, et al. (1996) restriction effective in preventing diabetes mellitus in the Otsuka Long Evans Substitution at Codon 269 (GlutamineRProline) of the Leptin Receptor (OB-R) Tokushima Fatty rat, a model of spontaneous non-insulin-dependent diabetes cDNA Is the Only Mutation Found in the Zucker Fatty (fa/fa) Rat. Biochemical mellitus? Diabetes Research and Clinical Practice 27: 97–106. and Biophysical Research Communications 224: 597–604. 54. Reaven GM (2005) The Insulin Resistance Syndrome: Definition and Dietary 31. Phillips MS, Liu Q, Hammond HA, Dugan V, Hey PJ, et al. (1996) Leptin Approaches to Treatment. Annu Rev Nutr 25: 391–406. receptor missense mutation in the fatty Zucker rat. Nat Genet 13: 18–19. 55. Stevens J, Green M, Kaiser D, Pohl S (1981) Insulin resistance in adipocytes 32. Takaya K, Ogawa Y, Isse N, Okazaki T, Satoh N, et al. (1996) Molecular from fed and fasted obese rats: Dissociation of two insulin actions. Molecular and Cloning of Rat Leptin Receptor Isoform Complementary DNAs–Identification Cellular Biochemistry 37: 177–183.

PLoS ONE | www.plosone.org 8 June 2011 | Volume 6 | Issue 6 | e21342